Pharmaceutical compositions containing functionalized triblock copolymers

ABSTRACT

Amphiphilic triblock copolymers B-A-B, wherein A is a linear poly(ethylene glycol) block, having a number average molecular weight (M n ) of between 900 and 3000 Daltons, determined with size exclusion chromatography; wherein B are hydrophobic blocks with at least two cyclic monomers selected from the group consisting of glycolide, lactide, 1,3-dioxan-2-one, 5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one, 1,4-dioxepan-2-one, 1,5-dioxepan-2-one, each B-block having a number average molecular weight (M n ) of between 400 and 2000 Daltons, determined with size exclusion chromatography; and wherein 25% to 100% of the polymer hydroxyl end-groups are covalently modified with at least one derivative of a C 2 -C 20  fatty acid. The invention also relates to compositions with such polymers and the use thereof.

FIELD OF THE INVENTION

The present invention relates to amphiphilic triblock copolymers,compositions comprising the copolymers and at least one therapeuticallyactive agent as well as implants comprising the copolymers.

BACKGROUND OF THE INVENTION

Controlled release of therapeutically active agents has become essentialin treatments of humans and animals.

In recent years, a number of polymers fabricated into devices asmicrospheres, microcapsules, liposomes, strands and the like have beendeveloped for this reason. The active agent is incorporated into theinterior of the devices and is after administration to the human oranimal body slowly released by different mechanisms. U.S. Pat. Nos.4,079,038, 4,093,709, 4,131,648, 4,138,344, 4,180,646, 4,304,767,4,946,931 and 5,968,543 disclose various types of polymers that may beused for the controlled delivery of active agents. The fabrication ofsuch devices is in many cases cumbersome, expensive and may also sufferfrom irreproducibility in the release kinetics. Furthermore, in mostcases an organic solvent is used which may have adverse effect on thetherapeutic agent and there could also be residual solvent in thedevice, which in many cases is highly toxic. Moreover the administrationof the solution or dispersion containing the devices is not patientfriendly, due to the high viscosity of such solutions or dispersions.Further, such devices are not generally useful for the delivery ofproteins that usually undergo a loss of activity during theirincorporation into the solid polymer.

An important improvement was found in the use of amphiphilic copolymers,especially triblock copolymers BAB with poly(ethylene glycol) as thecentral hydrophilic block A and terminal hydrophobic blocks B, withpolymer hydroxyl end-groups modified with fatty acid derivatives. Suchcopolymers may form micelles or thermo-reversible gels in aqueoussolutions that may contain at least one therapeutically active agent.

Micelles of the amphiphilic copolymer have a number of usefulattributes. For example when micelles having the correct size are used,which is usually below 40 nm, they will not extravasate in normalvasculature, but are able to extravasate in a tumor that normally has aleaky vasculature. Because of this it is possible to achieve a highconcentration of anti-neoplastic agents in the tumor, without incurringexcessive toxicity in normal tissues.

In addition to the usefulness as micelles in tumor targeting, micellesalso find important applications in the solubilisation of highly waterinsoluble drugs, since such drugs may be incorporated in the hydrophobiccore of the micelle.

The use of micelles in tumor targeting and solubilisation of highlywater-insoluble drugs has been extensively described by V. P. Torchilin,“Structure and design of polymeric surfactant-based drug deliverysystems”, J. Controlled Release 73 (2001) 137-172, and by V. P.Torchilin, “Polymeric Immunomicelles: Carriers of choice for targeteddelivery of water-insoluble pharmaceuticals”, Drug Delivery Technology,4 (2004) 30-39.

Micelles based on poly(ethylene glycol) and poly(D,L-lactic acid) havebeen investigated by J. Lee, “Incorporation and release behavior ofhydrophobic drug in functionalized poly(D,L-lactide)-block poly(ethyleneoxide) micelles” J. Controlled Release, 94 (2004) 323-335. Micellesbased on poly(ethylene glycol) and poly(β-benzyl-L-aspartate) have beeninvestigated by Kataoka, G. Kwon, “Block copolymer micelles for drugdelivery: loading and release of doxorubicin” J. Controlled Release, 48(1997) 195-201. Micelles based on poly(ethylene glycol) and poly(orthoester) have been described by Toncheva et. al., “Use of block copolymersof poly(ortho esters) and poly(ethylene glycol) micellar carriers aspotential tumor targeting systems”, J. Drug Targeting, 11 (2003)345-353.

It is also possible for the amphiphilic copolymers of the invention toform a so-called thermo-reversible gel in an aqueous solution. Such acopolymer solution has the peculiar property that at room temperature itis water-soluble and at the body temperature of 37° C. it becomeswater-insoluble and forms a gel.

The composition containing the copolymer and the therapeutically activeagent may be administered at room temperature as a low viscosity aqueoussolution, using a small gauge needle, thus minimizing discomfort for thepatient. Once at body temperature the composition will form awell-defined gel that will be localized at the desired site within thebody. Further, such materials are also uniquely suited for use with aprotein as the therapeutically active agent since the protein is simplydissolved in the same solution that contains the amphiphilic copolymerand the solution is injected, without affecting the properties of theprotein.

The therapeutically active agent is slowly released by diffusion, or bya combination of diffusion and erosion, from the micelles or thethermogels made of amphiphilic copolymers. Ultimately, the amphiphiliccopolymer has to fall apart into small fragments that can be metabolizedor removed from the body.

Thermogels have been extensively investigated. The most extensivelyinvestigated thermo gelling polymer is poly(N-isopropyl acrylamide).This polymer is soluble in water below 32° C. and sharply precipitatesas the temperature is raised above 32° C. This temperature is known asthe lower critical solution temperature, or LCST. Thus, such a polymercould be injected at room temperature as a low viscosity solution usinga small bore needle, and once in the tissues, it would precipitate,forming a well-defined depot. However, such polymers are non-degradable.Such polymers were extensively described by Hoffman, in L. C. Dong et.al., “Thermally reversible hydrogels: III. Immobilization of enzymes forfeedback reaction control”, J. Controlled Release, 4 (1986) 223-227.

Thermogels using poly(lactide-co-glycolide) copolymers as thehydrophobic segment and poly(ethylene glycol) as the hydrophilic segmenthave been extensively investigated and are described in a number ofpatents and publications: U.S. Pat. Nos. 5,702,717, 6,004,573,6,117,949, 6,201,072 B1. G. Zentner, J. Controlled Release, 72 (2001)203-215.

Thermogels using poly(L-lactide-co-ε-caprolactone) copolymers as thehydrophobic segment en poly(ethylene glycol) as the hydrophilic segmenthave been described in US 2007/0265356. This patent describes end groupmodification with aliphatic hydrocarbons, in particular C₃-C₁₈ aliphatichydrocarbons.

In an article published in Angew. Chem. Int. Ed. 2006, 45, p 2232-2235,“A Subtle End-Group Effect on Macroscopic Physical Gelation of TriblockCopolymer Aqueous Solutions”, BAB blockcopolymers having the blocksPLGA/PEG/PLGA are described. The PEG (i.e. polyethylene glycol A-block)is viewed as the hydrophilic block, the PLGA (i.e. poly(lacticacid-co-glycolic acid B-block) is the hydrophobic block. The articleshows that end-groups to the BAB block are important. If the end-groupis a hydrogen atom, a soluble system is prepared. If the end-groups areacetate or propionate a thermo reversible gel can be prepared (which gelexists at room temperature, i.e. 25° C.). If the end-groups arebutyrate, the modified blockcopolymer precipitates in a region from 0°C. to 50° C. The extent of esterification (i.e. endcapping in thecontext of the mentioned article) was higher than 90% for allderivatives.

A disadvantage of triblock copolymers known in the prior art, is that itis difficult to obtain an optimal balance between the polymer'shydrophilicity and hydrophobicity while at least maintainingbiodegradability. It is therefore difficult to obtain polymers with agood water solubility and the ability to retain (hydrophobic)therapeutically active agents.

Another disadvantage of triblock copolymers known in the prior art, isthat the thermogels formed at body temperature are only able to delivertherapeutically active agents for a few days except very hydrophobicdrugs like paclitaxel, due to very fast diffusion of the drug out of thegel mass.

Another disadvantage of triblock copolymers known in the prior art is,that the biodegradability is either very fast (in the order of days) orvery slow (in the order of months). This makes these copolymers lesssuitable for controlled drug release applications in which a treatmentin the order of a week or a few weeks, especially when the controlledrelease is largely determined by the degradation (erosion) of the gelinstead of diffusion of the medicament out of the gel (which may be thecase for very hydrophobic drugs)

SUMMARY OF THE INVENTION

It is an object of the present invention to provide triblock copolymers,which offer a variety of conditions which broaden the scope oftherapeutically active agents which can be delivered in a controlledmanner and which copolymers enable tuning the time required to degradein the human or animal body. It is also an object of the presentinvention to provide triblock copolymers of which are biodegradable.

DETAILED DESCRIPTION OF THE INVENTION

This object is achieved by providing an amphiphilic triblock copolymerB-A-B, wherein A is a linear poly(ethylene glycol) block, having anumber average molecular weight (M_(n)) of between 500 and 3000 Daltons,determined with size exclusion chromatography; wherein B are hydrophobicblocks comprising at least two cyclic monomers selected from the groupconsisting of glycolide, lactide, ε-caprolactone, p-dioxanone(1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one),1,4-dioxepan-2-one (including its dimer1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomorpholine, pivalolactone,chi.-diethylpropiolactone, ethylene carbonate, ethylene oxalate,3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxan-2,5-dione,6,8-dioxabicycloctane-7-one, ε-propiolactone, γ-butyrolactone,δ-valerolactone, ε-decalactone, 3-methyl-1,4-dioxane-2,5-dione,1,4-dioxane-2,5-dione, 2,5-diketomorpholine, α,α-diethylpropiolactone,γ-butyrolactone, 1,4-dioxepan-2-one, 1,5-dioxepan-2-one,6,6-dimethyl-dioxepan-2-one, 6,8-dioxabicycloctane-7-one,5,5-dimethyl-1,3-dioxan-2-one, each B-block having a number averagemolecular weight (M_(n)) of between 400 and 3000 Daltons, determinedwith size exclusion chromatography; and wherein 25% to 100% of thepolymer hydroxyl end-groups are covalently modified with at least onederivative of a C₂-C₂₀ fatty acid and wherein the B-block does notinclude the combination of glycolide and lactide and not the combinationof lactide and ε-caprolactone.

In one embodiment the invention relates to an amphiphilic triblockcopolymer B-A-B, wherein A is a linear poly(ethylene glycol) block,having a number average molecular weight (M_(n)) of between 900 and 3000Daltons, determined with size exclusion chromatography; wherein B arehydrophobic blocks comprising at least two cyclic monomers selected fromthe group consisting of glycolide, lactide, ε-caprolactone,1,3-dioxan-2-one, 5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,1,4-dioxepan-2-one, 1,5-dioxepan-2-one, each B-block having a numberaverage molecular weight (M_(n)) of between 400 and 2000 Daltons,determined with size exclusion chromatography; and wherein 25% to 100%of the polymer hydroxyl end-groups are covalently modified with at leastone derivative of a C₂-C₂₀ fatty acid, and wherein the B-block does notinclude the combination of glycolide and lactide and not the combinationof lactide and ε-caprolactone.

The polymers of the present invention are designed to broaden the scopeof therapeutically active agents which can be delivered in a controlledmanner and to tune the time required to degrade in the human or animalbody in such a way that full degradation is obtained shortly after fulldrug release.

The block ratio, in the context of the present invention, is the ratiobetween the sum of the number average molecular weights (M_(n)) of bothhydrophobic blocks without counting the end group modification (the sumof the two B blocks) and the polyethylene glycol A-block.

The block ratio should be high enough to ensure that micelles or gelscan be formed when dissolving the triblock copolymers in aqueoussolutions, but low enough so that the copolymers do not start toprecipitate in these aqueous solutions.

The required block ratio also depends on the hydrophobic blockcomposition (i.e. B-blocks), and the degree of modification and natureof the fatty acid derivative used for end group modification.

Solubility of the triblock copolymers is tightly linked to thehydrophobicity of the polyester blocks. The more hydrophobic thepolyester is, the lower the block ratio may be.

End group modification also influences the solubility of the triblockcopolymers according to the present invention. Longer fatty acids willrender the triblock copolymers more hydrophobic, and as a result, theblock ratio will have to decrease to maintain solubility in aqueoussolutions. The degree of modification of end-groups will also affectsolubility. A triblock according to the present invention modified at100% with a fatty acid will be more hydrophobic than the same triblockcopolymer modified at 50% with the same fatty acid, so the block ratiowill have to be lower with the fully modified copolymer to reach thesame solubility in aqueous solutions.

In an embodiment, the block ratio, which is defined as the ratio betweenthe sum of the number average molecular weight of the B-blocks and thenumber average molecular weight of the A-block, ranges between 0.5 and3, preferably between 0.5 and 1.7, more preferably between 0.6 and 1.5,even more preferably between 0.7 and 1.3.

A-Block

The A-block in the triblock copolymer may be a linear poly(ethyleneglycol) with a number average molecular weight which ranges between 500and 3000 Daltons, or between 900 and 2500 Daltons.

Poly(ethylene glycol) is a diol also known as poly(ethylene oxide) andboth names can be used interchangeably for the purpose of thisinvention.

B-Block

The B-blocks in the triblock copolymer may be hydrophobic blocks made byring-opening polymerization of 2 or more cyclic monomers and with anumber average molecular weight ranges between 400 and 3000 Daltons.Preferably the number average molecular weight of each B-block rangesbetween 450 and 2000 Dalton, more preferably between 500 and 1500Dalton.

Cyclic monomers used to make B blocks are selected from the groupconsisting of glycolide, lactide, ε-caprolactone, p-dioxanone(1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one),1,4-dioxepan-2-one (including its dimer1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomorpholine, pivalolactone,chi.-diethylpropiolactone, ethylene carbonate, ethylene oxalate,3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxan-2,5-dione,6,8-dioxabicycloctane-7-one, β-propiolactone, γ-butyrolactone,δ-valerolactone, ε-decalactone, 3-methyl-1,4-dioxane-2,5-dione,1,4-dioxane-2,5-dione, 2,5-diketomorpholine, α,α-diethylpropiolactone,γ-butyrolactone, 1,4-dioxepan-2-one, 1,5-dioxepan-2-one,6,6-dimethyl-dioxepan-2-one, 6,8-dioxabicycloctane-7-one,5,5-dimethyl-1,3-dioxan-2-one, or preferably of the group consisting ofglycolide, lactide, ε-caprolactone, 1,3-dioxan-2-one (also known astrimethylene carbonate), 5,5-dimethyl-1,3-dioxan-2-one,1,4-dioxan-2-one, 1,4-dioxepan-2-one, 1,5-dioxepan-2-one, and whereinthe B-block does not include the combination of glycolide and lactideand not the combination of lactide and ε-caprolactone.

Hydrophobic blocks containing the monomeric units described above mainlycontain ester and/or carbonate bonds, making them easily biodegradable.They can be prepared in a range of well-defined molecular weights. Thisenables the fabrication of triblock copolymers that have a well-definedstructure, so that well-defined micelles or thermogels can be formedfrom the copolymers and moreover good reproducibility in the releasekinetics of the therapeutically active agent may be achieved.

The choice of monomers is based primarily on the rate and profile ofbiodegradation that one wants to achieve with the triblock copolymer invivo. Polyesters made by combining these monomers have been studied fora while and some of the combinations are well known.

In most cases, the combinations involve only 2 monomers, although thereare examples with 3 monomers in rare cases.

Biodegradation in the context of the present invention is assessed bythe macroscopic disappearance of the polymer under its shape in the body(gel, thermogel, micelles).

The degradation of the polyesters blocks down to monomeric residues isnot something that can be easily followed in vivo, and it usually takeslonger to occur. It can be assessed in vitro by various analyticaltechniques including size-exclusion chromatography, nuclear magneticresonance, MALDI-TOF, high pressure liquid chromatography andcombinations of those.

The polyester combinations described below are chosen based ontheoretical degradation in vitro. The biodegradation in vivo willusually be faster since a simple hydrolysis of the ester bond betweenthe polyester blocks and the polyethylene glycol block will result in asevere disturbance of the macroscopic state of the polymer (gel,thermogel, micelle).

In an embodiment B-blocks comprise monomer combinations comprisingbetween 50 and 100 mol % glycolide. Such B-blocks will be among thefastest to biodegrade. Preferably B-blocks comprise between 60 and 95mol % glycolide, more preferably between 75 and 90 mol %. Combinationsof glycolide with other monomers will result in tunablebiodegradability. For example the time to degrade will increase in therange glycolide-lactide, glycolide-trimethylene carbonate andglycolide-caprolacton.

In an embodiment B-blocks comprise monomer combinations includingbetween 50 and 100 mol %, preferably between 60 and 95 mol %, morepreferably between 75 and 90 mol % lactide. Such combinations will alsodegrade relatively fast, but slower than the ones with equivalentamounts of glycolide. The time to degrade will also depend on whetherracemic lactide or L-lactide is used. The higher crystallinity ofL-lactide usually yields polyesters which take longer to degrade, longerthan with racemic lactide. Such polymers will take a few weeks todegrade.

In an embodiment B-blocks comprise monomer combinations comprisingbetween 50 and 100 mol %, preferably between 60 and 95 mol %, morepreferably between 75 and 90 mol % trimethylene carbonate. Suchcombinations usually exhibit very slow biodegradation with the exceptionof lactide-trimethylene carbonate and glycolide-trimethylene carbonate.The resulting polyesters also contain carbonate bonds, giving them anamorphous state which tends to favor erosion-based biodegradation overbulk biodegradation, prolonging the macroscopic state of a gel,thermogel or micelle when compared with polymers degrading through bulkerosion (typically the ones based on lactide-glycolide polyesters). Inthe case of drug delivery, this makes it easier to control the deliveryrate. These polyesters take at least 3 months to degrade, and triblockcopolymers made from these at least 2 months.

In an embodiment B-blocks comprise monomer combinations comprisingbetween 50 and 100 mol %, preferably between 60 and 95 mol %, morepreferably between 75 and 90 mol % 5,5-dimethyl-1,3-dioxan-2-one (alsocalled 5,5-dimethyl trimethylene carbonate) Such combinations willexhibit even slower degradation than with trimethylene carbonate, whilestill providing polyesters containing carbonate bonds to have amorphousproperties, beneficial for erosion-based biodegradation. Thesepolyesters take at least 4 months to degrade, and triblock copolymersmade from these at least 3 months.

Other combinations of the listed monomers are also possible, and theskilled person is able to choose them according to the polymerproperties that they need for a specific application.

The hydrophobic-type monomers of the B-blocks can be categorized intogroups according to relative degree of hydrophobicity. Relatively lowhydrophobicity monomers are for example 1,4-dioxan-2-one, glycolide,1,5-dioxepane-2-one. Relatively high hydrophobicity monomers includelactide, ε-caprolactone and 5,5-dimethyl-1,3-dioxan-2-one. In the case atriblock copolymer is desired which has a slow degradation profile,monomers are selected that have a rather high hydrophobicity andoptionally the B-block has a higher molecular weight. In the case atriblock copolymer is desired which has a fast degradation profile, theB blocks are built from monomers having a low hydrophobicity(hydrophilic monomers).

In an embodiment one of the cyclic monomers of the B-blocks is selectedfrom the group consisting of glycolide, lactide, ε-caprolactone and1,3-dioxan-2-one. Preferably, one of the cyclic monomers of the B-blocksis lactide or ε-caprolactone. Preferred combinations of cyclic monomersin the B-blocks of the copolymers according to the present inventioninclude but are not limited to:

-   -   glycolide and a monomer of the group of 1,3-dioxan-2-one,        5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,        1,4-dioxepan-2-one, 1,5-dioxepan-2-one.    -   lactide and a monomer of the group of 1,3-dioxan-2-one,        5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,        1,4-dioxepan-2-one, 1,5-dioxepan-2-one.    -   1,3-dioxan-2-one and a monomer of the group of        5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,        1,4-dioxepan-2-one, 1,5-dioxepan-2-one.    -   ε-caprolactone and a monomer of the group of 1,3-dioxan-2-one,        5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,        1,4-dioxepan-2-one, 1,5-dioxepan-2-one.

Preparation of B-A-B Triblock Copolymers

B-A-B triblock copolymers may be synthesized by ring-openingpolymerization, or polycondensation reactions.

B blocks can be polymerized by using the cyclic monomers mentioned abovein a ring-opening polymerization using the hydroxyl end-groups ofpoly(ethylene glycol) to initiate the polymerization. This is a verycontrolled and straightforward way of preparing triblocks in one stepfor people skilled in the art. Schemes and details for similarring-opening polymerization reactions can be found in several patents orpatent applications including and not limited to EP0863745 andWO0018821.

An alternative is to prepare B blocks separately by using ring-openingpolymerization initiated with a short mono functional alcohol, and thencoupling these B blocks with poly(ethylene glycol) in the presence ofcoupling agents like isocyanates. Coupling reactions may also be doneafter activation of functional end-groups with activating agents likecarbonyl diimidazole, N-hydroxysuccinimide, para-nitrophenylchloroformate, succinic anhydride and the like.

Preparing B blocks by polycondensation reactions using the open form ofthe cyclic monomers mentioned above, such as lactic acid, glycolic acid,epsilon-hydroxyhexanoic acid and the like is also possible.Nevertheless, obtaining well-defined blocks in terms of averagemolecular weight and end group functionality with polycondensationreactions is particularly difficult, even for someone skilled in theart.

Thus obtained triblock copolymers usually have hydroxyl moieties at bothextremities. Without modification, only very specific triblocks willform thermo-reversible gels or micelles in aqueous solutions. In thepresent invention, these hydroxyl moieties are therefore modified withderivatives of natural-occurring fatty acids to achieve formation ofgels or micelles while keeping the polymer water-soluble andbiodegradable.

End Group Modification of B-A-B Triblock Copolymers

B-A-B triblocks are preferably partially or completely modified usingthe terminal hydroxyl group of the B blocks. Fatty acids include aselection ranging from 2 to 20, preferably 6-18 carbons, saturated orunsaturated, preferably with even numbers of carbons. Most fatty acidswith an odd number of carbons are not naturally present in warm bodiesand thus less desirable from a polymer biodegradation point of view.Fatty acids with more than 20 carbons are very hydrophobic solids, andyields water-insoluble polymers when used in the scope of thisinvention.

Preferably, the fatty acids derivatives used to modify the polymerhydroxyl end-groups are selected from the group consisting ofderivatives of caproic acid, caprylic acid, capric acid, lauric acid,myristic acid, myristoleic acid, palmitic acid, palmitoleic acid,stearic acid, oleic acid, linoleic acid, alpha-linoleic acid,gamma-linoleic acid, stearidonic acid, rumenic acid, beta-calendic acid,eleostearic acid, puninic acid, parinaric acid, pinolenic acid,arachidic acid, eicosenoic acid, eicosadienoic acid, eicosatrienoicacid, dihomo-gamma-linolenic acid, mead acid, eicosatetraenoic acid,arachidonic acid, eicosapentaenoic acid.

These naturally occurring fatty acids are easily degradable through theacetyl-coenzyme A cycle. Furthermore these acids have less risk ofexhibiting toxicity in vivo in quantities used in the scope in thepresent invention. Some of them could have beneficial or detrimentalbiological activities though. A person skilled in the art would have totake the fatty acid choice into account, depending on the applicationand the location in the body.

Derivatives of fatty acids refer to fatty acids which may have beenmodified or activated to allow coupling reactions with the triblockcopolymers hydroxyl end-groups.

Coupling fatty acids to the B-A-B triblock copolymers may involve theuse of coupling agents like (but not limited to) isocyanates or thederivatisation of either the fatty acids or the polymer end-groups.Functional groups of the fatty acids or polymers can be activated topromote coupling by using activating agents like (but not limited to)carbonyl diimidazole, N-hydroxysuccinimide, para-nitrophenylchloroformate, succinic anhydride. Direct derivatives of fatty acidslike but not limited to acid chlorides, anhydrides, isocyanates can alsobe used, especially since some of them are readily commerciallyavailable.

These coupling methods are well known to the one skilled in the art.

In one embodiment of the invention terminal hydroxyl groups of the Bblocks are modified with fatty acids having between 2 and 6 carbonatoms.

The degree of modification of the polymers hydroxyl end-groups is anumerical value which quantifies the percentage of hydroxyl end-groupsthat have been modified with fatty acids derivatives. A degree ofmodification of 100% means that both polymer extremities have beenentirely modified. 50% means that half of the extremities (one out oftwo) have been modified. This value, as well as the triblock averagemolecular weight, is preferably calculated using nuclear magneticresonance, since it is one of the few analytical methods giving accessto absolute numerical values, as opposed to analytical methods likesize-exclusion chromatography, where the average-molecular weight is avalue which is relative to a polymer standard such as polystyrene.

The optimal degree of modification which makes the polymers in thispresent invention able to form micelles or thermogels in aqueoussolutions is dependent on various factors such as triblock averagemolecular weight, block ratio, monomer composition, nature of the fattyacids derivatives.

The hydrophobicity of the triblock copolymers according to the presentinvention will increase when the fatty acid derivatives is longer, forthe same degree of end group modification.

The hydrophobicity of the triblock copolymers according to the presentinvention will increase when the degree of end-capping (i.e. end groupmodification) increases, for the same fatty acid derivatives.

To achieve solubility in aqueous solutions at a certain polymerconcentration, as well as specific molecular assembly such as micellesor (thermo)gels, the fatty acid and the degree of end group modificationshould be chosen and tuned together with block length, block ratio andpolyester block composition.

Modification with longer fatty acid derivatives will generally increasethe degradation time of the polymer.

A triblock copolymer according to the present invention has two OHend-groups, which may be modified. A statistical distribution ofmolecules having 0, 1 or 2 modified end-groups will result in a degreeof modification other then 100%, for example 60%. Various polymerpurification methods can allow a person skilled in the art to narrowthis distribution of polymer chains by separating polymer chains whichare not modified (0%), half modified (50%) or completely modified(100%). The issue is that these purification methods are time-consumingand often not applicable to polymer batches larger than 5 grams. Havingpolymers with degrees of modification other than 0, 50 or 100% can benecessary to achieve proper preparation of gels or micelles depending onthe fatty acid derivative, the monomer composition and the polymer blockratio and the polymer block size.

In the scope of the present invention, the numerical range for thedegree of modification is between 25 and 100%, preferably between 40%and 98%, more preferably between 50 and 95%. The ranges are derived fromexperimental results with various fatty acids, monomer composition,block ratio and block size.

Triblock copolymers with degree of modification lower than 25% have beenfound to contain too much unmodified polymer chains to form thermogelsin the solubility range that they have in aqueous solutions.

In a preferred embodiment at least 90% of the polymer hydroxylend-groups of a polymer according to the present invention arecovalently modified with at least one derivative of a C₂-C₂₀ fatty acid.

In this embodiment the degree of end group modification is thus at least90%.

Such modified triblock copolymers provide a well defined structure andeasily fold into U-shapes which assemble to form micelles withhydrophobic cores and hydrophilic shells in aqueous solutions.

In an embodiment, the block copolymer with the general formula B-A-Bcomprises poly(ethylene glycol) (PEG) as A-block, having a numberaverage molecular weight of between 1000 and 2500 Dalton, preferablybetween 1100 and 2000 Dalton, determined with Size ExclusionChromatography (SEC). The B-blocks are hydrophobic blocks comprising atleast two cyclic monomers selected from the group consisting ofglycolide, lactide, 1,3-dioxan-2-one (also known as trimethylenecarbonate), 5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,1,4-dioxepan-2-one, 1,5-dioxepan-2-one, each B-block having a numberaverage molecular weight of between 400 and 1600 Dalton, preferablybetween 500 and 1500, more preferably between 600 and 1300 Dalton,determined with Size Exclusion Chromatography (SEC), wherein the B-blockdoes not include the combination of glycolide and lactide.

In one embodiment the invention relates to an amphiphilic triblockcopolymer B-A-B, comprising poly(ethylene glycol) (PEG) as A-blockhaving a number average molecular weight of between 1000 and 2500Dalton, determined with Size Exclusion Chromatography (SEC); theB-blocks being hydrophobic blocks comprising at least two cyclicmonomers selected from the group consisting of glycolide, lactide,1,3-dioxan-2-one, 5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,1,4-dioxepan-2-one, 1,5-dioxepan-2-one, each B-block having a numberaverage molecular weight of between 400 and 2500, determined with SizeExclusion Chromatography (SEC); wherein the amphiphilic triblockcopolymer has a block ratio, defined as the ratio between the sum of thenumber average molecular weight of the B-blocks and the number averagemolecular weight of the A-block, of between 0.5 and 2.5; and wherein 25%to 100% of the hydroxyl endgroups are covalently modified with at leastone derivative of a C₂-C₂₀ fatty acid derivatives, the fatty acids beingselected from the group consisting of derivatives of acetic acid,butyric acid, caproic acid, caprylic acid, capric acid, lauric acid,myristic acid, myristoleic acid, palmitic acid, palmitoleic acid,stearic acid, oleic acid, linoleic acid, alpha-linoleic acid,gamma-linoleic acid, stearidonic acid, rumenic acid, beta-calendic acid,eleostearic acid, puninic acid, parinaric acid, pinolenic acid,arachidic acid, eicosenoic acid, eicosadienoic acid, eicosatrienoicacid, dihomo-gamma-linolenic acid, mead acid, eicosatetraenoic acid,arachidonic acid, eicosapentaenoic acid; and wherein the B-block doesnot include the combination of glycolide and lactide.

The block ratio, defined as the ratio between the sum of the numberaverage molecular weight of the B-blocks and the number averagemolecular weight of the A-block, ranges between 0.5 and 3, or between0.5 and 2.5, preferably between 0.6 and 2.2, more preferably between 0.7and 1.7. In this embodiment 25% to 100% of the hydroxyl endgroups arecovalently modified with at least one derivative of a C₂-C₂₀, preferablyC₆-C₁₈ fatty acid derivatives. In this embodiment, the fatty acidsderivatives used to modify the polymer hydroxyl end-groups arepreferably selected from the group consisting of derivatives of aceticacid, butyric acid, caproic acid, caprylic acid, capric acid, lauricacid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid,stearic acid, oleic acid, linoleic acid, alpha-linoleic acid,gamma-linoleic acid, stearidonic acid, rumenic acid, beta-calendic acid,eleostearic acid, puninic acid, parinaric acid, pinolenic acid,arachidic acid, eicosenoic acid, eicosadienoic acid, eicosatrienoicacid, dihomo-gamma-linolenic acid, mead acid, eicosatetraenoic acid,arachidonic acid, eicosapentaenoic acid.

Biodegradation in the context of the present invention refers to thedegradation, disassembly, digestion or disappearance of the amphiphiliccopolymers by action of the biological environment, including action ofliving organisms and most notably at physiological pH and temperature. Aprincipal mechanism for biodegradation in the present invention is thehydrolysis of linkages between and within the monomer units of theamphiphilic copolymers. Specific reactions include but are not limitedto ester hydrolysis (chemical or enzymatic) and degradation of fattyacid fragments via lipolysis or oxidation.

Polymers of the present invention may be solubilised in aqueoussolutions with concentrations preferably ranges between 3 and 50% byweight of the polymer. The most preferable concentrations to achievethermogelation of formation of micelles are dependent on the polymercomposition. Addition of therapeutically active agents to a polymersolution usually affects the optimal concentration to form micelles orthermogels, wherever the agents are dissolved, emulsified or suspended.

The invention also relates to compositions comprising at least oneamphiphilic triblock copolymer of the present invention and a medicallyaccepted solvent. A medically accepted solvent may be for example water;a mixture of water and an organic solvent like for example ethanol,isopropanol and DMSO; an isotonic aqueous solution which is suitable forinjection in the human or animal body (i.e. in the context of thepresent invention a solution having an osmotic pressure comparable or atleast compatible with the osmotic pressure of human or animal bodilyfluids, like blood); benzylbenzoate and isopropyl myristate.

In an embodiment, such a composition comprises at least onetherapeutically active agent and is a pharmaceutical composition.

By therapeutically active agents people skilled in the art refer to anyset of molecules, cells or cell materials able to prevent, slow down,moderate or cure a disease in, or that can deliver a desired therapeuticeffect on, a treated human or animal. Human diseases are referred to asdefined by the World Health Organization in the WHO ICD-10 (2007)classification document.

Therapeutically active agents include but are not limited to nutrients,pharmaceuticals (small molecular entities), proteins and peptides,vaccines, genetic materials, (such as polynucleotides, oligonucleotides,plasmids, DNA and RNA), diagnostic agents, imaging agents, enzymes,nucleic acid sequences, antigens, antibodies, antibody fragments,viruses, virus-based materials, cells, cell substructures, growthfactors, antibiotics, anti-inflammatory compounds, immune-modulating,anti-thrombogenic compounds, anti-claudicating drugs, anti-arrhythmicdrugs, anti-atherosclerotic drugs, antihistamines, cancer drugs,vascular drugs, ophthalmic drugs, amino acids, vitamins, hormones,neurotransmitters, neurohormones, enzymes, signaling molecules,psychoactive medicaments, synthetic drugs, semi-synthetic drugs, naturaldrugs and substances derived from these, or combinations of the above.

The active pharmaceutical ingredient (API), may demonstrate any kind ofactivity, depending on the intended use. The active agent may be capableof stimulating, blocking or suppressing a biological response.

The therapeutic active agents can be used for sustained delivery in manydifferent diseases and conditions within humans and animal.

In an embodiment the therapeutically active agent is a growth factor.Such a composition is very suitable for application in orthopedics andin particular in the prevention or treatment of diseases ofintervertebral discs. This is because the composition will gel and holdthe active agent in place over a period in time, releasing it in acontrolled manner than straight injection of a non-gelling solution.Furthermore, the gel-forming polymers will be completely broken downafter having completed their function. This is especially important inthe application in the area of intervertebral discs, where there is lessmetabolic activity.

Preferably as growth factor at least one compound is used of the groupconsisting of transforming growth factor beta-3, osteogenic protein 1,bone morphogenic protein 2 and 7. Although less preferred it is alsopossible to use compositions containing thermogels in general and atransforming growth factor. Such a composition at least has theadvantage of the slow release of the growth factor.

In yet another embodiment the therapeutic active agent is an agent tosuppress or slow down cancerous growth or neovascularisation, such asanti-VEGF agents, si-RNA or aptamers.

In still another embodiment the therapeutic active agent is an agent toavoid, control, suppress, or eradicate infectious diseases.

The copolymers of the present invention will find utility in any of theuses for which biodegradable polymers are useful, including such uses asvehicles for the sustained and controlled release of therapeuticallyactive agents, implants, tissue-engineering devices, and the like, theywill also find particular utility in applications where their nature asblock copolymers having both hydrophilic and hydrophobic segmentsconfers a special benefit, and those uses will be addressed in greaterdetail below.

For some applications special moieties may have to be introduced intothe fatty acid derivatives used for end group modification. For example,the use of unsaturated fatty acid may allow chemical reactions to occurbetween the unsaturated fatty acid chains to achieve polymercrosslinking. Crosslinking is usually carried out in order to modify themechanical properties and degradation profile of polymers. Theactivation and intermolecular reaction between those crosslinkablemoieties is usually caused by a radiation source, an external chemicalreaction or stimulus, or a combination thereof. Radiation examplesinclude, but are not limited, to heat, infrared sources, ultra-violetsources, electron-beam sources, micro-waves sources, x-ray sources,visible light sources [monochromatic or not] and gamma-rays. Externalreaction, or stimulus include, but are not limited, to pH,oxidation/reduction reactions, reactions with a chemical agent presentin vivo (gas, protein, enzymes, antibody etc), reaction with a chemicaladded to the composition upon introduction into the body, known as dualsystems, for example a molecule containing two or more reactive groups.

Micellar Systems.

In one preferred embodiment a composition according to the present, themedically accepted solvent comprises water and the copolymer is presentin a concentration above its critical micelle concentration (CMC), suchthat micelles are formed in an aqueous solution, the therapeuticallyactive agent being entrapped in or controlled released by the micelles.

When the copolymers are placed in water, in which the hydrophilicsegment is soluble and the hydrophobic segment is insoluble, the polymerchains may spontaneously self-aggregate to form micellar structuresdepending on their concentration.

One major utility of such micellar structures resides in their abilityto entrap, controlled release and or solubilise hydrophobic drugs in thehydrophobic core of micelles. Such retention can be carried out in anumber of ways. The drug may be added to the aqueous media containingthe micelles and incorporated by simple stirring, by heating to moderatetemperatures or by ultrasonification or by active loading as used inliposome production processes. Alternately, a drug dissolved in avolatile organic solvent is added to a water solution of preformedmicelles with a subsequent solvent evaporation from the system.

While any of the anticancer agents that can be incorporated in micellarstructures are suitable for this use, anticancer agents that areparticularly suitable for micellar tumor targeting are those with lowwater solubility such as doxorubicin, daunorubicin, epirubicin,mitomicin C, paclitaxel, cis-platin, carboplatin, and the like. Otheragents may include anticancer proteins such as neocarzinostatin,L-aspariginase, and the like and photosensitizers used in photodynamictherapy.

In addition to the usefulness as micelles in tumor targeting, micellesalso find important applications in the solubilisation of highly waterinsoluble drugs, since such drugs may be incorporated in the hydrophobiccore of the micelle.

Thermogels.

In another preferred embodiment a composition according to the presentinvention, the medically accepted solvent comprises water and thecomposition, being an aqueous solution, has a lower critical solutiontemperature (LCST) of between 4 and 37° C., such that the aqueoussolution undergoes a sol-gel transition starting between 4 and 37° C.

Preferably, the composition also contains a therapeutically activeagent.

The solution according to this embodiment has a lower critical solutiontemperature (LCST) below the warm body temperature (37° C. for a humanbody for example).

Such polymers are water-soluble below their LCST, also known as geltemperature, due to strong hydrogen bonding between the hydrophilic partof the chains and water, but above the LCST, hydrogen interactions areweakened and hydrophobic interactions between the hydrophobic domains ofthe polymer become dominant with consequent precipitation of thepolymer, which can result in gelation of the polymer solution.

The LCST value depends on the balance of hydrophilic and hydrophobicportions of the block copolymer and can be adjusted by varying thisbalance. It also depends on the concentration of the block copolymer inthe aqueous solution. Materials having particular usefulness fortherapeutic applications are those where the LCST value is between 20and warm body temperature since such materials will be soluble inaqueous solutions at room temperature and form a gel at body temperature(37° C. for a human body for example).

One of the desirable features of thermogels is the ability to administerthermogel formulations using a small bore needle resulting insignificantly less pain on administration relative to the administrationof microspheres, microcapsules, strands, or other solid drug-releasingdevices. This is due to the water solubility of thermogels at roomtemperature, and the relatively low viscosity of the aqueous solutionmaking the use of small-bore needles possible.

Another important and unique feature is the ability to delivertherapeutically active agents at a controlled rate and without loss ofbiological activity. In this application, the polymer according to theinvention can be dissolved in an appropriate volume of an aqueoussolution and the peptide, protein or nucleic acid sequence is dissolvedin the same solution. The mixture is then injected in the desired bodysite, where it gels, entrapping the peptide, protein or nucleic acidsequence in the gelled material. It will be appreciated that these areextremely mild conditions since active agents are only exposed to waterand at temperatures no higher than the warm body temperature.

This method is greatly superior to conventional methods of biomoleculeincorporation into solid polymers that require harsh conditions such aselevated temperatures, and/or organic solvents, or mixtures of organicsolvents and water and or surfactants, which usually results in loss ofprotein activity.

This method is particularly useful for the delivery and dosing oftherapeutically active agents in applications including but not limitedto injections of the thermogels containing the biomolecules mentionedabove into articulate cartilage, pericardium, cardiac muscles, scleraand the vitreous body of the eye.

The LCST behavior also gives advantages when building composite devices.They can be built by using several thermogels with different LCST(always below warm body temperature). Upon implantation the in vitrodegradation and release of actives can be tuned depending on their LCSTand chemical structures.

The present invention further relates to applications of amphiphilictriblock copolymers according to the present invention and compositionsthereof. In particular the present invention relates to medical devicescomprising compositions comprising at least one amphiphilic triblockcopolymer according to the present invention.

Medical Devices

Bio Erodible Copolymer. Matrix for Controlled Delivery and TissueEngineering

The invention also relates to an implant containing the polymeraccording to the invention. In certain uses it is desirable to have amaterial that has improved mechanical properties relative to thermogelling materials. To this effect, solid polymers can be prepared thatare useful in a number of applications, for example orthopedicapplications such as fracture fixation, or repair of osteochondraldefects and the like. The solid polymer can be readily fabricated into anumber of shapes and forms for implantation, insertion or placement onthe body or into body cavities or passageways. For example, the blockcopolymer of this invention may be injection-molded, extruded orcompression-molded into a thin film, or made into devices of variousgeometric shapes or forms such as flat, square, round, cylindrical,tubular, discs, rings and the like. Rod, or pellet-shaped devices may beimplanted using a trocar, and these, or other shapes, may be implantedby minor surgical procedures. Alternatively, a device may be implantedfollowing a major surgical procedure such as tumor removal in thesurgical treatment of cancer. The implantation of polymer waferscontaining anticancer agents is described for example, in Brem et. al.,U.S. Pat. Nos. 5,626,862 and 5,651,986 and references cited therein; andthe block and graft copolymers will find utility in such applications.

Tissue Engineering

Applications of tissue engineering devices comprising thermogels madewith copolymers according to the present invention include but are notlimited to nerve growth or repair, cartilage growth or repair, bonegrowth or repair, muscle growth or repair, skin growth or repair,secreting gland repair, ophtalmic repair. It should be underlined thatthermogels may be used as such or as a part of a bigger implant,scaffold or structure.

Thermogel formulations with LCST below warm body temperatures may alsobe used as temporary void fillers in case of significant trauma, toprevent adhesion of damage tissues and scar tissue formation whilewaiting for corrective and reconstructive surgery. Void filling could beperformed easily by injecting the thermogel formulation and removalcould be performed via cutting, scraping or suction after cooling downthe area to liquefy the thermogel. Other benefits of using void fillersmay include but are not limited to: preventing contamination fromoutside, preventing infection, preventing surrounding tissue necrosis oralteration, inducing specific tissue formation (bone, cartilage, muscle,nerve, skin etc.), helping to maintain structural integrity of thesurrounding tissues by itself or by combination with other knownscaffolds or structures, trapping specific natural or foreign molecules.

Measurement Methods

The number average and weight average molecular weights (M_(n) andM_(w), respectively) of the triblock copolymers are determined with SizeExclusion Chromatography (SEC). Size-Exclusion Chromatography isperformed with an Agilent 1100 series machine equipped with athermostatically controlled double-C column system, in tetrahydrofuranat 25° C. Detection is done by refractive index measurement and UV. 50micrograms of polymer solutions at 1 mg/ml are injected and runs lastedabout 30 minutes. The external standards are series of polystyrenepolymers. Relative values of the number average molecular weight M_(n)and the weight average molecular weight M_(w) can be obtained, as wellas the polydispersity. The unit Daltons is equivalent to g/mol.

The molecular structure is determined with proton and carbon nuclearmagnetic resonance (¹H NMR and ¹³C NMR, respectively), using deuteratedchloroform (chloroform-d3) as solvent and reference.

Nuclear magnetic resonance is performed with a Brücker NMR Advance 300(300 MHz) using chloroform-d3 as a solvent. Dimethylsulfoxide-d6 anddeuterium oxide can be used in specific cases when polymer solubility inchloroform is too low. Samples concentration is about 10 mg/ml for aproton spectrum measurement and 20-30 mg/ml for a carbon spectrummeasurement. For carbon measurement, DEPT135 measurements were alsoperformed to differentiate carbon types. From the integration of variousproton signals, absolute number average molecular weights M_(n) can beobtained.

LCST properties (T₁) (loss modulus G′, storage modulus G″, and complexviscosity η of the copolymers as a function of temperature) aredetermined by rheology (oscillation mode) using a Physica MC 301 (AntonPaar) rheometer. Rheological properties at increasing temperatures weredetermined using the same polymer concentration as that used in gellingexperiments, usually 20 wt %. Viscosity (y-axis, in Pa·s) was plottedversus temperature (x-axis, in ° C.). Although rheological measurementsactually determined the onset of gelation shown as an increase ofviscosity as a function of temperature, we defined the LCST as thetemperature at which the viscosity started to increase.

T₂ is determined by heating a gelled composition and visually determinewhen the thermogelling polymer precipitates.

Intrinsic viscosities are measured using a cone-plate rheometer inrotation mode at various temperatures, using a Physica MC 301 (AntonPaar) rheometer

Gelation tests are carried out in 12 mm diameter glass tubes. Acopolymer of this invention is dissolved at 20° C. in 10 mM phosphatebuffered saline (PBS) at pH 7.4, at a 15 wt % concentration. 1 mL ofpolymer solution is transferred into a test tube and it is closed with asilicon cap. Then the test tube is placed into a thermostaticallycontrolled water bath at 37° C. After predetermined intervals of time(e.g. 15 min, 30 min and 2 hours), the tube is taken out and turnedupside down for 15 seconds. Gelation is considered complete when thepolymer solution does not flow at all during 15 seconds. This test isqualitative and used for fast screening of polymers. It does not provideaccurate values for LCST and gel mechanical properties.

Degradation time of the B-blocks, the triblock copolymers or materialsin general can be assessed in vitro by various analytical techniquesincluding size-exclusion chromatography, nuclear magnetic resonance,MALDI-TOF, high pressure liquid chromatography and combinations ofthose. The degradation experiments are carried out in 12 mm diameterglass tubes with volume markings. The copolymer is dissolved at 20° C.in 10 mM phosphate buffered saline (PBS) at pH 7.4, and at a 20 wt %concentration. 3.0 mL of solution is poured into each tube to ensure asolid gelation. The glass tubes are placed in a thermostaticallycontrolled bath for 30 minutes to make the 3 mL solutions gel. Then, 7.0ml of 10 mM PBS at pH 7.4 incubated at the same temperature were placedover the gels. At predetermined time periods, the buffer over the gelwas withdrawn and the remaining volume of gel was measured through thevolume marking. Then 7.0 mL of fresh buffer pre-incubated at the sametemperature were added and the tubes were placed again into thethermostatically controlled bath. The remaining gel volumes were plottedagainst incubation time to get the degradation profiles. Atpredetermined amounts of time, pieces of gels can also be removed andanalysed by NMR and SEC to calculate the decrease of the number averagemolecular weight over time.

The invention is explained in detail with the following examples:

EXAMPLE 1 Triblock Polymerization with L-lactide and 1,3-dioxan-2-one

In a 500 mL 2 necks round-bottom flask equipped with magnetic stirring,polyethyleneglycol (50.0 g, 33.3 mmol) was dissolved in 250 mL of drytoluene (<60 μg H₂O per liter) at room temperature. Using a Dean-Starkdevice with a cooler on top, 150 mL of toluene were distilled off toremove water azeotropically by heating at 140° C. at atmosphericpressure.

After cooling down the solution at 100° C., L-lactide (30.0 g, 208 mmol)and 1,3-dioxan-2-one (30.0 g, 294 mmol) were added at once via thesecond neck of the flask and 50 mL of dry toluene were added to cleanthe neck. Using the Dean-Stark device and the cooler again, 50 mL oftoluene were distilled off to remove water from the monomers by heatingat 140° C. at atmospheric pressure. 100 mL of dry toluene were left inthe flask for the polymerization.

After cooling down the mixture at 100° C., Tin(II) 2-ethylhexanoate(0.50 g, ˜0.5 wt % versus monomers) was added through the second neck,the Dean-Stark device was removed and the cooler placed directly on topof the flask.

Then the polymerization was done at reflux (120° C.) for a predeterminedamount of time (from 16 h to 3 days).

After cooling down at room temperature, the polymer solution wastransferred into a one liter round-bottom flask equipped with a powerfulmagnetic stirring system. 800 mL of dry diethyl ether were slowly addedunder vigorous stirring (1000 rpm) to make the polymer phase separate asan oil. After 10 minutes of decantation, the top phase (toluene, ether,unreacted monomers, catalyst) was removed by pouring. 20 mL of drymethylene chloride were added to make the polymer less viscous and then400 mL of dry diethyl ether were added under vigorous stirring (1000rpm) to wash the polymer. After 10 minutes of decantation, the top phase(methylene chloride, ether and impurities) was removed by pouring it. Asecond washing with 400 mL of dry diethyl ether after addition of 20 mLdry methylene chloride to the polymer was performed. After decantation,the top phase was removed, and the concentrated polymer was dried at 60°C. under vacuum (20 mbar) for 2 hours in a rotavapor.

Drying of the polymer was completed at room temperature in a drying ovenwith phosphorous pentoxide at 30° C. and under vacuum (50 m bar) for 3days.

Then the polymer looked like a colourless transparent paste. Thetriblock copolymer was characterized by proton nuclear magneticresonance in deuterated chloroform and size-exclusion chromatography intetrahydrofuran (double C-column system).

A triblock copolymer of this example had B-blocks comprising 41 mol %L-lactide and 59 mol % 1,3-dioxan-2-one. Each B-block has a numberaverage molecular weight of around 700 Daltons. The number averagemolecular weight of the polyethylene glycol A-block is around 1500Daltons. The block ratio therefore is around 1.2.

EXAMPLE 2 Very Hydrophobic Polymer: Triblock Polymerization withL-lactide and 5,5-dimethyl-1,3-dioxan-2-one

In a 500 mL 2 necks round-bottom flask equipped with magnetic stirring,polyethyleneglycol (50.0 g, 33.3 mmol) were dissolved in 250 mL of drytoluene (<60 μg H₂O per liter) at room temperature. Using a Dean-Starkdevice with a cooler on top, 150 mL of toluene were distilled off toremove water azeotropically by heating at 140° C. at atmosphericpressure.

After cooling down the solution at 100° C., L-lactide (25.0 g, 173 mmol)and 5,5-dimethyl-1,3-dioxan-2-one (25.0 g, 192 mmol) were added at oncevia the second neck of the flask and 50 mL of dry toluene were added toclean the neck. Using the Dean-Stark device and the cooler again, 50 mLof toluene were distilled off to remove water from the monomers byheating at 140° C. at atmospheric pressure. 100 mL of dry toluene wereleft in the flask for the polymerization.

After cooling down the mixture at 100° C., Tin(II) 2-ethylhexanoate(0.50 g, ˜0.5 wt % versus monomers) was added through the second neck,the Dean-Stark device was removed and the cooler placed directly on topof the flask.

Then the polymerization was done at reflux (120° C.) for 3 days.

After cooling down at room temperature, the polymer solution wastransferred into a one liter round-bottom flask equipped with a powerfulmagnetic stirring system. 800 mL of dry diethyl ether were slowly addedunder vigorous stirring (1000 rpm) to make the polymer phase separate asan oil. After 10 minutes of decantation, the top phase (toluene, ether,unreacted monomers, catalyst) was removed by pouring. 20 mL of drymethylene chloride were added to make the polymer less viscous and then400 mL of dry diethyl ether were added under vigorous stirring (1000rpm) to wash the polymer. After 10 minutes of decantation, the top phase(methylene chloride, ether and impurities) was removed by pouring it. Asecond washing with 400 mL of dry diethyl ether after addition of 20 mLdry methylene chloride to the polymer was performed. After decantation,the top phase was removed, and the concentrated polymer was dried at 60°C. under vacuum (20 mbar) for 2 hours in a rotavapor.

Drying of the polymer was completed at room temperature in a drying ovenwith phosphorous pentoxide at 30° C. and under vacuum (50 mbar) for 3days.

Then the polymer looked like a slightly yellow paste. The triblockcopolymer was characterized by proton nuclear magnetic resonance indeuterated chloroform and size-exclusion chromatography intetrahydrofuran (double C-column system).

A triblock copolymer of this example had B-blocks comprising 45 mol %L-lactide and 55 mol % 5,5-dimethyl-1,3-dioxan-2-one. Each B-block had anumber average molecular weight of around 750 Daltons. The numberaverage molecular weight of the polyethylene glycol A-block was around1500 Daltons. The block ratio therefore was around 1.0.

EXAMPLE 3 40 to 50% Modification of the Triblock Copolymer of Example 1With A) Octanoyl Chloride (Caprylic Acid Derivative) or B) AceticAnhydride

In a 250 mL round-bottom flask equipped with magnetic stirring, thetriblock from example 1 (30.0 g) was heated at 60° C. and connected to ahigh vacuum pump for drying at 0.1 mbar for 2 hours with slow magneticstirring. After cooling at room temperature, the flask was flushed withnitrogen and then 80 mL dry methylene chloride were added via a syringe,followed by the addition of triethylamine (1.1 equivalent per polymermole). Then a dropping funnel was mounted above the flask and filledwith octanoyl chloride (1.0 equivalent per polymer mole for a maximum of50% modification) diluted 5 times with dry methylene chloride.

The polymer solution was cooled down at 10° C., stirred at 400 rpm andoctanoyl chloride was added in 30 minutes. After addition, the droppingfunnel was removed and the flask was flushed with nitrogen again. Thecold bath was removed and the reaction was allowed to run at roomtemperature overnight and followed by nuclear magnetic resonance untilthe desired degree of modification was achieved.

After reaction, solvents and unreacted triethylamine were removed usinga rotavapor (45° C., 20 mbar) for one hour. Then the residue wasredissolved using 200 mL of dry ethyl acetate to make thetriethylammonium salt precipitate. The precipitate was removed using aglass filter (pore size 4) filled with Celite® filter agent. Then 100 mLethyl acetate were removed using a rotavapor (40° C., 20 mbar) for 10minutes.

To the concentrated polymer solution were added 300 mL of dry pentaneunder vigorous stirring (1000 rpm) to make the polymer phase separateand to remove unreacted octanoyl chloride and caprylic (i.e. octanoic)acid. After decantation, the solvent phase was removed by pouring it outof the flask. 20 mL were added to make the polymer less viscous and then200 mL of dry pentane were added under vigorous stirring (1000 rpm) towash the polymer. After decantation and removal of the solvent phase(upper phase), 20 mL were added to make the polymer less viscous andthen 200 mL of dry pentane were added under vigorous stirring (1000 rpm)to wash the polymer a second time. After decantation and removal of thesolvent phase (upper phase), the concentrated polymer was dried at 50°C. under vacuum (20 mbar) for 2 hours in a rotavapor.

Drying of the polymer was completed at room temperature in a drying ovenwith phosphorous pentoxide at 30° C. and under vacuum (50 mbar) for 2days. The modified triblock copolymer was characterized by protonnuclear magnetic resonance in deuterated chloroform and size-exclusionchromatography in tetrahydrofuran (double C-column system).

The procedure for modification with acetic anhydride is different fromthe procedure described above. However, modification with aceticanhydride is commonly known in the art.

In the case of the example 1 triblock copolymer, 50% modification withoctanoyl chloride (C₈), example 4A, gave practically the same result interms of aqueous solubility and gelation as the as a 80% modificationwith acetic anhydride (C₂), example 4B.

EXAMPLE 4 Modification of the Triblock Copolymer of Example 2 With A)Butanoyl Chloride or B) Acetic Anhydride

The triblock copolymer of example 2 is modified in a similar way asdescribed in example 4 at 25-30% with acetic anhydride (C₂) or butanoylchloride (C₄) to yield a hydrophobic gel with a degradation time,determined of longer than a month.

EXAMPLE 5 Preparation of Paclitaxel-Loaded Micelles

The triblock copolymer from example 1 and Paclitaxel (1:0.4 w/w) weredissolved in acetonitrile and thoroughly mixed. The solvent wasevaporated using a stream of nitrogen under stirring. The mixture wasre-dissolved in distilled water and a solution with strong opalescencewas obtained. After filtration (G3 filter), the solution waslyophilized. Micelles containing Paclitaxel could be smoothlyre-dissolved in water and characterized by light-scatteringmeasurements.

EXAMPLE 6 In Vitro Release of Bovine Serum Albumine (BSA) from aThermogel Followed by UV-Visible Light Spectroscopy

The release experiments were carried out in 12 mm diameter glass tubes.The copolymer was dissolved at 20° C. in 10 mM phosphate buffered saline(PBS) at pH 7.4 at a 15 wt % concentration. BSA at a loading of 1 wt %and 5 wt % was dissolved in the same buffer and mixed with the copolymersolution.

The glass tubes were placed in an incubator with a shaking bath at 37°C. or in a water-bath thermostatically controlled at 37° C. for 1 hour.The dimensions of the gel were 20 mm high×12 mm diameter. Then, 2 ml of10 mM PBS at pH 7.4 or 2 ml incubated at the same temperature wereplaced over the gels. At predetermined time periods, the buffer over thegel was withdrawn and replaced with a fresh buffer pre-incubated at thesame temperature. The withdrawn samples were analyzed by UV-visiblelight spectroscopy using the absorption at 494 nm for pH 7.4 and theabsorption at 458 nm for pH 5.5.

EXAMPLE 7 Use of Thermogels as Temporary Void Filler and Shock Absorberin a Maxillo-Facial Trauma

Upon arrival of a patient to the emergency ward, and after diagnosis ofa significant maxillo-facial trauma, a biodegradable thermogel would beinjected in the damage areas in order to relieve pain (via an analgesiccontained in the composition) and act as a shock absorber between brokenbone and tissue parts upon gelation. The gel would also prevent unwantedadhesion of damaged tissue and bones to prevent scar tissue formation.This would give the surgeons more time to plan reconstructive surgeryand would cause less trauma for the patient during reconstructivesurgery because spontaneous healing would be delayed for a few days. Bythe time the surgeons would be ready, the gel would have starteddegrading or remaining gel blocks could be removed by cooling them downusing cold fluids or instruments and then by sucking the liquefied gelout.

EXAMPLE 8 Injection of a Thermogel Containing Osteogenic and/or BoneMorphogenic Proteins into Intervertebral Discs or Articulate Cartilageto Stop or Reverse Degeneration of Diseased or Damaged Tissues

A composition of the thermogel with a LCST of 37° C. containing amongstother components the growth factor TGF-beta-3, or another osteogenic orbone morphogenic protein was prepared. The composition in its liquidform was injected into the intervertebral disc using a small bore needleor a small diameter cannula. Upon reaching LCST, the composition wouldgel and hold the growth factor in situ over a period of time, releasingit in a slower manner than straight injection of a non-gelling solution.

What is claimed is:
 1. A pharmaceutical composition, comprising: anamphiphilic triblock copolymer B-A-B, a medically accepted solvent andan therapeutically active agent, wherein A is a linear poly(ethyleneglycol) block having a number average molecular weight (M_(n)) ofbetween 500 and 3000 Daltons, determined with size exclusionchromatography; wherein B are hydrophobic blocks comprising at least twocyclic monomers of glycolide, lactide, ε-caprolactone, p-dioxanone(1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one),1,4-dioxepan-2-one (including its dimer1,5,8,12-tetraoxacyclo-tetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomorpholine, pivalolactone,ε-diethylpropiolactone, ethylene carbonate, ethylene oxalate,3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxan-2,5-dione,6,8-dioxabicycloctane-7-one, β-propiolactone, γ-butyrolactone,δ-valerolactone, ε-decalactone, 3-methyl-1,4-dioxane-2,5-dione,1,4-dioxane-2,5-dione, 2,5-diketo-morpholine, α,α-diethylpropiolactone,γ-butyrolactone, 1,4-dioxepan-2-one, 1,5-dioxepan-2-one,6,6-dimethyl-dioxepan-2-one, 6,8-dioxabicycloctane-7-one, or5,5-dimethyl-1,3-dioxan-2-one, each B-block having a number averagemolecular weight (M_(n)) of between 400 and 3000 Daltons, determinedwith size exclusion chromatography; and wherein 25% to 100% of thepolymer hydroxyl end-groups are covalently modified with at least oneacid chloride, anhydride or isocyanate derivative of a C₂-C₂₀ fattyacid, and wherein the B-blocks comprise monomer combinations comprisinga) between 50 and 100 mol % 5,5-dimethyl-1,3-dioxan-2-one (also called5,5-dimethyl trimethylene carbonate), b) glycolide and at least onemonomer that is 1,3-dioxan-2-one, 5,5-dimethyl-1,3-dioxan-2-one,1,4-dioxan-2-one, 1,4-dioxepan-2-one, or 1,5-dioxepan-2-one, c) lactideand at least one monomer that is 1,3-dioxan-2-one,5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one, 1,4-dioxepan-2-one, or1,5-dioxepan-2-one, or d) 1,3-dioxan-2-one and at least one monomer thatis 5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one, 1,4-dioxepan-2-one,or 1,5-dioxepan-2-one.
 2. The pharmaceutical composition according toclaim 1, wherein a block ratio, which is defined as a ratio between asum of the number average molecular weight of the B-blocks and thenumber average molecular weight of the A-block, ranges between 0.5 and3.
 3. The pharmaceutical composition according to claim 2, wherein thenumber average molecular weight (M_(n)) of the linear poly(ethyleneglycol) block ranges between 900 and 3000 Daltons; wherein the numberaverage molecular weight (M_(n)) of each B-block ranges between 400 and2000 Daltons; wherein the fatty acids derivatives used to modify thepolymer hydroxyl end-groups are derivatives of caproic acid, caprylicacid, capric acid, lauric acid, myristic acid, myristoleic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleicacid, alpha-linoleic acid, gamma-linoleic acid, stearidonic acid,rumenic acid, beta-calendic acid, eleostearic acid, puninic acid,parinaric acid, pinolenic acid, arachidic acid, eicosenoic acid,eicosadienoic acid, eicosatrienoic acid, dihomo-gamma-linolenic acid,mead acid, eicosatetraenoic acid, arachidonic acid, or eicosapentaenoicacid; and wherein at least 90% of the polymer hydroxyl end-groups arecovalently modified with the at least one acid chloride, anhydride orisocyanate derivative of a C₂-C₂₀ fatty acid.
 4. A pharmaceuticalcomposition according to claim 3, wherein one of the cyclic monomers ofthe B-blocks is glycolide, lactide, ε-caprolactone or 1,3-dioxan-2-one.5. A pharmaceutical composition, comprising: an amphiphilic triblockcopolymer B-A-B, a medically accepted solvent and an therapeuticallyactive agent, wherein A is a linear poly(ethylene glycol) block having anumber average molecular weight (M_(n)) of between 500 and 3000 Daltons,determined with size exclusion chromatography; wherein B are hydrophobicblocks comprising at least two cyclic monomers of glycolide, lactide,ε-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylene carbonate(1,3-dioxan-2-one), 1,4-dioxepan-2-one (including its dimer1,5,8,12-tetraoxacyclo-tetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomorpholine, pivalolactone,ε-diethylpropionlactone, ethylene carbonate, ethylene oxalate,3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxan-2,5-dione,6,8-dioxabicycloctane-7-one, β-propiolactone, γ-butyrolactone,δ-valerolactone, ε-decalactone, 3-methyl-1,4-dioxane-2,5-dione,1,4-dioxane-2,5-dione, 2,5-diketo-morpholine, α,α-diethylpropiolactone,γ-butyrolactone, 1,4-dioxepan-2-one, 1,5-dioxepan-2-one,6,6-dimethyl-dioxepan-2-one, 6,8-dioxabicycloctane-7-one or5,5-dimethyl-1,3-dioxan-2-one, each B-block having a number averagemolecular weight (M_(n)) of between 400 and 3000 Daltons, determinedwith size exclusion chromatography; and wherein 25% to 100% of thepolymer hydroxyl end-groups are covalently modified with at least oneacid chloride, anhydride or isocyanate derivative of a C₂-C₂₀ fatty,acid and wherein the B-blocks comprise monomer combinations a)comprising ε-caprolactone and at least one monomer that is1,3-dioxan-2-one, 5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,1,4-dioxepan-2-one, or 1,5-dioxepan-2-one, b) consisting of between 50and 100 mol % glycolide; and caprolactone as a second monomer, or c)having between 50 and 100 mol % trimethylene carbonate.
 6. Thepharmaceutical composition according to claim 5, wherein a block ratio,which is defined as a ratio between a sum of the number averagemolecular weight of the B-blocks and the number average molecular weightof the A-block, ranges between 0.5 and
 3. 7. The pharmaceuticalcomposition according to claim 6, wherein the number average molecularweight (M_(n)) of the linear poly(ethylene glycol) block ranges between900 and 3000 Daltons and the number average molecular weight (M_(n)) ofeach B-block ranges between 400 and 2000 Daltons; wherein the fattyacids derivatives used to modify the polymer hydroxyl end-groups arederivatives of caproic acid, caprylic acid, capric acid, lauric acid,myristic acid, myristoleic acid, palmitic acid, palmitoleic acid,stearic acid, oleic acid, linoleic acid, alpha-linoleic acid,gamma-linoleic acid, stearidonic acid, rumenic acid, beta-calendic acid,eleostearic acid, puninic acid, parinaric acid, pinolenic acid,arachidic acid, eicosenoic acid, eicosadienoic acid, eicosatrienoicacid, dihomo-gamma-linolenic acid, mead acid, eicosatetraenoic acid,arachidonic acid or eicosapentaenoic acid; and wherein at least 90% ofthe polymer hydroxyl end-groups are covalently modified with the atleast one acid chloride, anhydride or isocyanate derivative of a C₂-C₂₀fatty acid.
 8. The pharmaceutical composition according to claim 1,wherein the B-blocks comprise the monomer combinations comprising a)between 50 and 100 mol % 5,5-dimethyl-1,3-dioxan-2-one (also called5,5-dimethyl trimethylene carbonate).
 9. The pharmaceutical compositionaccording to claim 1, wherein the B-blocks comprise the monomercombinations comprising the b) glycolide and at least one monomer thatis 1,3-dioxan-2-one, 5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one,1,4-dioxepan-2-one, or 1,5-dioxepan-2-one.
 10. The pharmaceuticalcomposition according to claim 1, wherein the B-blocks comprise themonomer combinations comprising the c) lactide and at least one monomerthat is 1,3-dioxan-2-one, 5,5-dimethyl-1,3-dioxan-2-one,1,4-dioxan-2-one, 1,4-dioxepan-2-one, or 1,5-dioxepan-2-one.
 11. Thepharmaceutical composition according to claim 1, wherein the B-blockscomprise the monomer combinations comprising the d) 1,3-dioxan-2-one andat least one monomer that is 5,5-dimethyl-1,3-dioxan-2-one,1,4-dioxan-2-one, 1,4-dioxepan-2-one, or 1,5-dioxepan-2-one.
 12. Thepharmaceutical composition according to claim 5, wherein the B-blockscomprise the monomer combinations a) comprising ε-caprolactone and atleast one monomer that is 1,3-dioxan-2-one,5,5-dimethyl-1,3-dioxan-2-one, 1,4-dioxan-2-one, 1,4-dioxepan-2-one, or1,5-dioxepan-2-one.
 13. The pharmaceutical composition according toclaim 5, wherein the B-blocks comprise the monomer combinations b)consisting of between 50 and 100 mol % glycolide; and caprolactone as asecond monomer.
 14. The pharmaceutical composition according to claim 5,wherein the B-blocks comprise the monomer combinations c) having between50 and 100 mol % trimethylene carbonate.